Methods of making gene expression libraries

ABSTRACT

Provided herein are methods of determining a location of a target nucleic acid in a biological sample.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationSer. No. 62/980,867, filed Feb. 24, 2020; the entire contents of whichare herein incorporated by reference.

BACKGROUND

Cells within a tissue of a subject have differences in cell morphologyand/or function due to varied analyte levels (e.g., gene and/or proteinexpression) within the different cells. The specific position of a cellwithin a tissue (e.g., the cell's position relative to neighboring cellsor the cell's position relative to the tissue microenvironment) canaffect, e.g., the cell's morphology, differentiation, fate, viability,proliferation, behavior, and signaling and cross-talk with other cellsin the tissue.

Spatial heterogeneity has been previously studied using techniques thatonly provide data for a small handful of analytes in the context of anintact tissue or a portion of a tissue, or provide a lot of analyte datafor single cells, but fail to provide information regarding the positionof the single cell in a parent biological sample (e.g., tissue sample).

SUMMARY

This application is based on the discovery of a method of making aspatial 5′ gene expression library for spatial analysis of targetanalytes, including long target analytes e.g., VDJ rearranged T-cellreceptors or immunoglobulins.

Provided herein are methods of identifying a location of a targetnucleic acid in a permeabilized biological sample, the methodcomprising: (a) generating a cDNA molecule comprising a sequence that issubstantially complementary to the target nucleic acid using a reversetranscription primer comprising (i) a sequence that is substantiallycomplementary to a portion of the target nucleic acid and (ii) a firstadaptor sequence, wherein the step of generating the cDNA moleculeoccurs within the permeabilized biological sample; (b) ligating a secondadaptor sequence to a 3′ end of the cDNA molecule, wherein the step ofligating is performed within the biological sample; (c) after step (b),releasing the cDNA molecule from the target nucleic acid, such that thecDNA contacts an array, wherein the array comprises an attached captureprobe comprising in a 5′ to a 3′ direction: (i) a spatial barcode and(ii) a capture domain that binds specifically to the second adaptorsequence ligated to the cDNA; (d) after step (c), extending a 3′ end ofthe capture probe using the cDNA as a template; and (e) determining (i)all or a part of the sequence of the target nucleic acid, or acomplement thereof, and (ii) all or a part of the sequence of thespatial barcode, or a complement thereof, and using the determinedsequences of (i) and (ii) to identify the location of the target nucleicacid in the permeabilized biological sample.

Also provided herein are methods of identifying a location of a targetnucleic acid in a permeabilized biological sample, the methodcomprising: (a) generating a cDNA molecule comprising a sequence that issubstantially complementary to the target nucleic acid using a reversetranscription primer comprising (i) a sequence that is substantiallycomplementary to a portion of the target nucleic acid and (ii) a firstadaptor sequence, wherein the step of generating the cDNA moleculeoccurs within the permeabilized biological sample; (b) extending a 3′end of the cDNA molecule to include a second adaptor sequence, whereinthe step of extending is performed within the biological sample; (c)releasing the cDNA molecule from the target nucleic acid, such that thecDNA contacts an array, wherein the array comprises an attached captureprobe comprising in a 5′ to a 3′ direction: (i) a spatial barcode and(ii) a capture domain that binds specifically to the second adaptorsequence; (d) extending a 3′ end of the capture probe using the cDNA asa template; and (e) determining (i) all or a part of the sequence of thetarget nucleic acid, or a complement thereof, and (ii) the sequence ofthe spatial barcode, or a complement thereof, and using the determinedsequences of (i) and (ii) to identify the location of the target nucleicacid in the permeabilized biological sample. In some embodiments, step(b) can occur simultaneously with step (a).

In some embodiments of any of the methods described herein, steps (a)through (c) are performed when the biological sample is disposed on thearray.

In some embodiments of any of the methods described herein, step (a) isperformed when the biological sample is not disposed on the array andstep (b) is performed when the biological sample is disposed on thearray, and wherein the method further comprises between steps (a) and(b), a step of disposing the biological sample on the array.

In some embodiments of any of the methods described herein, steps (a)and (b) are performed when the biological sample is not disposed on thearray, and wherein the method further comprises between steps (b) and(c), a step of disposing the biological sample on the array.

In some embodiments of any of the methods described herein, the sequencethat is substantially complementary to a portion of the target nucleicacid present in the reverse transcription primer comprises a poly(T)sequence.

In some embodiments of any of the methods described herein, the sequencethat is substantially complementary to a portion of the target nucleicacid present in the reverse transcription primer comprises a randomsequence.

In some embodiments of any of the methods described herein, the secondadaptor sequence is a template switching oligonucleotide (TSO).

In some embodiments of any of the methods described herein, the arraycomprises a slide.

In some embodiments of any of the methods described herein, a 5′ end ofthe capture probe is attached to the slide.

In some embodiments of any of the methods described herein, the array isa bead array.

In some embodiments of any of the methods described herein, a 5′ end ofthe capture probe is attached to a bead of the bead array.

In some embodiments of any of the methods described herein, the captureprobe further comprises a unique molecular identifier (UMI).

In some embodiments of any of the methods described herein, the UMI ispositioned 5′ relative to the capture domain in the capture probe.

In some embodiments of any of the methods described herein, thedetermining in step (e) comprises sequencing (i) all or a part of thesequence of the target nucleic acid, or a complement thereof, and (ii)all or a part of the sequence of the spatial barcode, or a complementthereof.

In some embodiments of any of the methods described herein, thesequencing is high throughput sequencing.

In some embodiments of any of the methods described herein, thesequencing is sequencing by hybridization.

In some embodiments of any of the methods described herein, the targetnucleic acid is RNA.

In some embodiments of any of the methods described herein, the RNA isan mRNA.

In some embodiments of any of the methods described herein, thepermeabilized biological sample is a permeabilized tissue section.

In some embodiments of any of the methods described herein, thepermeabilized tissue section is a permeabilized formalin-fixed andparaffin-embedded (FFPE) tissue section.

Some embodiments of any of the methods described herein furthercomprises a step of imaging the biological sample.

In some embodiments of any of the methods described herein, the step ofimaging is performed prior to step (a).

In some embodiments of any of the methods described herein, the step ofimaging is performed between steps (b) and (c).

Some embodiments of any of the methods described herein furthercomprises, between steps (b) and (c), a step of freezing and thawing thepermeabilized biological sample.

Some embodiments of any of the methods described herein furthercomprises, between steps (b) and (c), a step of sectioning thepermeabilized biological sample.

In some embodiments of any of the methods described herein, the step ofsectioning the permeabilized biological sample is performed usingcryosectioning.

Some embodiments of any of the methods described herein furthercomprises, prior to step (a), a step of permeabilizing the biologicalsample.

In some embodiments of any of the methods described herein, theperformance of step (a) comprises introducing a reverse transcriptase,dNTPs, and the reverse transcription primer into the permeabilizedbiological sample.

In some aspects, provided herein are kits comprising: a reversetranscription primer comprising (i) a sequence that is substantiallycomplementary to a portion of the target nucleic acid and (ii) a firstadaptor sequence; a reverse transcriptase; and an oligonucleotidecomprising a second adaptor sequence or a complement thereof.

In some embodiments of any of the kits provided herein, the kit furthercomprises a ligase.

In some embodiments of any of the kits provided herein, the reversetranscriptase is a reverse transcriptase with terminal transferaseactivity.

In some embodiments of any of the kits provided herein, the secondadaptor sequence or the complement thereof is a TSO or a complementthereof.

In some embodiments of any of the kits provided herein, the kit furthercomprises an array, wherein the array comprises an attached captureprobe comprising in a 5′ to a 3′ direction: (i) a spatial barcode and(ii) a capture domain that binds specifically to the second adaptorsequence.

In another aspect, provided herein are nucleic acids comprisingcomprising, in the 5′ to 3′ direction: a spatial barcode; a sequencecomplementary to a second adaptor sequence; a sequence present in atarget nucleic acid; and a sequence complementary to a first adaptorsequence.

In another aspect, provided herein are nucleic acids comprising, in the3′ to 5′ direction: a complement of a spatial barcode; a second adaptorsequence; a sequence complementary to a sequence present in a targetnucleic acid; and a first adaptor sequence.

In some embodiments of any of the nucleic acids provided herein, thesecond adaptor sequence comprises a TSO.

In some embodiments of any of the nucleic acids provided herein, thesecond adaptor sequence comprises a complement of a TSO.

In some embodiments of any of the nucleic acids provided herein, thefirst adaptor sequence is a reverse transcriptase primer.

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, patent application, or item ofinformation was specifically and individually indicated to beincorporated by reference. To the extent publications, patents, patentapplications, and items of information incorporated by referencecontradict the disclosure contained in the specification, thespecification is intended to supersede and/or take precedence over anysuch contradictory material.

Where values are described in terms of ranges, it should be understoodthat the description includes the disclosure of all possible sub-rangeswithin such ranges, as well as specific numerical values that fallwithin such ranges irrespective of whether a specific numerical value orspecific sub-range is expressly stated.

The term “each,” when used in reference to a collection of items, isintended to identify an individual item in the collection but does notnecessarily refer to every item in the collection, unless expresslystated otherwise, or unless the context of the usage clearly indicatesotherwise. Various embodiments of the features of this disclosure aredescribed herein. However, it should be understood that such embodimentsare provided merely by way of example, and numerous variations, changes,and substitutions can occur to those skilled in the art withoutdeparting from the scope of this disclosure. It should also beunderstood that various alternatives to the specific embodimentsdescribed herein are also within the scope of this disclosure.

DESCRIPTION OF DRAWINGS

The following drawings illustrate certain embodiments of the featuresand advantages of this disclosure. These embodiments are not intended tolimit the scope of the appended claims in any manner. Like referencesymbols in the drawings indicate like elements.

FIG. 1A shows a workflow schematic illustrating exemplary, non-limitingsteps for in-situ cDNA synthesis and capturing.

FIG. 1B shows a workflow schematic illustrating exemplary, non-limitingsteps for building a 5′ spatial gene expression library.

FIG. 2A shows a workflow schematic illustrating exemplary, non-limitingsteps for in-situ cDNA synthesis and sample handling.

FIG. 2B shows a workflow schematic illustrating exemplary, non-limitingsteps for building a 5′ spatial gene expression library.

FIG. 3A shows a workflow schematic illustrating exemplary, non-limitingsteps for synthesizing a cDNA molecule.

FIG. 3B shows a workflow schematic illustrating exemplary, non-limitingsteps for cDNA binding to an attached capture probe and the extention ofthe capture probe using the cDNA molecule as a template, and thegeneration of a second strand complementary to the extended captureprobe.

FIG. 3C shows a workflow schematic illustrating exemplary, non-limiting,non-exhaustive steps for building a 5′ spatial gene expression library.

DETAILED DESCRIPTION

In some cases, spatial analysis methods can be carried out bypermeabilizing a biological sample, capturing analytes (e.g., nucleicacids (e.g., mRNA)) or intermediate agents on an array, and performingreverse transcription and sequencing steps to identify the location ofone or more analytes from the biological sample. Many capture protocolsrely on the use of a poly(A) tail, either natural or introduced. Severalchallenges can arise from these protocols. For example, there may or maynot be biased in the analytes or intermediate agents that are able tomigrate from a biologica sample to the array. As another example,capture by the poly(A) tail can lead to a 3′ bias in gene expressionlibraries generated from mRNAs due to limitations of some steps of theprocess. Provided herein are methods that can, in some cases, addressone or both of these challenges.

Provided herein are methods of identifying a location of a targetnucleic acid in a permeabilized biological sample, the methodcomprising: (a) generating a cDNA molecule comprising a sequence that issubstantially complementary to the target nucleic acid using a reversetranscription primer comprising (i) a sequence that is substantiallycomplementary to a portion of the target nucleic acid and (ii) a firstadaptor sequence, wherein the step of generating the cDNA moleculeoccurs within the permeabilized biological sample; (b) ligating a secondadaptor sequence to a 3′ end of the cDNA molecule, wherein the step ofligating is performed within the biological sample; (c) after step (b),releasing the cDNA molecule from the target nucleic acid, such that thecDNA contacts an array, wherein the array comprises an attached captureprobe comprising in a 5′ to a 3′ direction: (i) a spatial barcode and(ii) a capture domain that binds specifically to the second adaptorsequence ligated to the cDNA; (d) after step (c), extending a 3′ end ofthe capture probe using the cDNA as a template; and (e) determining (i)all or a part of the sequence of the target nucleic acid, or acomplement thereof, and (ii) all or a part of the sequence of thespatial barcode, or a complement thereof, and using the determinedsequences of (i) and (ii) to identify the location of the target nucleicacid in the permeabilized biological sample. Non-limiting aspects ofthese methods are described herein.

Spatial analysis methodologies and compositions described herein canprovide a vast amount of analyte and/or expression data for a variety ofanalytes within a biological sample at high spatial resolution, whileretaining native spatial context. Spatial analysis methods andcompositions can include, e.g., the use of a capture probe including aspatial barcode (e.g., a nucleic acid sequence that provides informationas to the location or position of an analyte within a cell or a tissuesample (e.g., mammalian cell or a mammalian tissue sample) and a capturedomain that is capable of binding to an analyte (e.g., a protein and/ora nucleic acid) produced by and/or present in a cell. Spatial analysismethods and compositions can also include the use of a capture probehaving a capture domain that captures an intermediate agent for indirectdetection of an analyte. For example, the intermediate agent can includea nucleic acid sequence (e.g., a barcode) associated with theintermediate agent. Detection of the intermediate agent is thereforeindicative of the analyte in the cell or tissue sample.

Non-limiting aspects of spatial analysis methodologies and compositionsare described in U.S. Pat. Nos. 10,774,374, 10,724,078, 10,480,022,10,059,990, 10,041,949, 10,002,316, 9,879,313, 9,783,841, 9,727,810,9,593,365, 8,951,726, 8,604,182, 7,709,198, U.S. Patent ApplicationPublication Nos. 2020/239946, 2020/080136, 2020/0277663, 2020/024641,2019/330617, 2019/264268, 2020/256867, 2020/224244, 2019/194709,2019/161796, 2019/085383, 2019/055594, 2018/216161, 2018/051322,2018/0245142, 2017/241911, 2017/089811, 2017/067096, 2017/029875,2017/0016053, 2016/108458, 2015/000854, 2013/171621, WO 2018/091676, WO2020/176788, Rodrigues et al., Science 363(6434):1463-1467, 2019; Lee etal., Nat. Protoc. 10(3):442-458, 2015; Trejo et al., PLoS ONE14(2):e0212031, 2019; Chen et al., Science 348(6233):aaa6090, 2015; Gaoet al., BMC Biol. 15:50, 2017; and Gupta et al., Nature Biotechnol.36:1197-1202, 2018; the Visium Spatial Gene Expression Reagent Kits UserGuide (e.g., Rev C, dated June 2020), and/or the Visium Spatial TissueOptimization Reagent Kits User Guide (e.g., Rev C, dated July 2020),both of which are available at the 10× Genomics Support Documentationwebsite, and can be used herein in any combination. Further non-limitingaspects of spatial analysis methodologies and compositions are describedherein.

Some general terminology that may be used in this disclosure can befound in Section (I)(b) of WO 2020/176788 and/or U.S. Patent ApplicationPublication No. 2020/0277663. Typically, a “barcode” is a label, oridentifier, that conveys or is capable of conveying information (e.g.,information about an analyte in a sample, a bead, and/or a captureprobe). A barcode can be part of an analyte, or independent of ananalyte. A barcode can be attached to an analyte. A particular barcodecan be unique relative to other barcodes. For the purpose of thisdisclosure, an “analyte” can include any biological substance,structure, moiety, or component to be analyzed. The term “target” cansimilarly refer to an analyte of interest.

Analytes can be broadly classified into one of two groups: nucleic acidanalytes, and non-nucleic acid analytes. Examples of non-nucleic acidanalytes include, but are not limited to, lipids, carbohydrates,peptides, proteins, glycoproteins (N-linked or O-linked), lipoproteins,phosphoproteins, specific phosphorylated or acetylated variants ofproteins, amidation variants of proteins, hydroxylation variants ofproteins, methylation variants of proteins, ubiquitylation variants ofproteins, sulfation variants of proteins, viral proteins (e.g., viralcapsid, viral envelope, viral coat, viral accessory, viralglycoproteins, viral spike, etc.), extracellular and intracellularproteins, antibodies, and antigen binding fragments. In someembodiments, the analyte(s) can be localized to subcellular location(s),including, for example, organelles, e.g., mitochondria, Golgi apparatus,endoplasmic reticulum, chloroplasts, endocytic vesicles, exocyticvesicles, vacuoles, lysosomes, etc. In some embodiments, analyte(s) canbe peptides or proteins, including without limitation antibodies andenzymes. Additional examples of analytes can be found in Section (I)(c)of WO 2020/176788 and/or U.S. Patent Application Publication No.2020/0277663. In some embodiments, an analyte can be detectedindirectly, such as through detection of an intermediate agent, forexample, a ligation product or an analyte capture agent (e.g., anoligonucleotide-conjugated antibody), such as those described herein.

A “biological sample” is typically obtained from the subject foranalysis using any of a variety of techniques including, but not limitedto, biopsy, surgery, and laser capture microscopy (LCM), and generallyincludes cells and/or other biological material from the subject. Insome embodiments, a biological sample can be a tissue section. In someembodiments, a biological sample can be a fixed and/or stainedbiological sample (e.g., a fixed and/or stained tissue section).Non-limiting examples of stains include histological stains (e.g.,hematoxylin and/or eosin) and immunological stains (e.g., fluorescentstains). In some embodiments, a biological sample (e.g., a fixed and/orstained biological sample) can be imaged. Biological samples are alsodescribed in Section (I)(d) of WO 2020/176788 and/or U.S. PatentApplication Publication No. 2020/0277663.

In some embodiments, a biological sample is permeabilized with one ormore permeabilization reagents. For example, permeabilization of abiological sample can facilitate analyte capture. Exemplarypermeabilization agents and conditions are described in Section(I)(d)(ii)(13) or the Exemplary Embodiments Section of WO 2020/176788and/or U.S. Patent Application Publication No. 2020/0277663.

Array-based spatial analysis methods involve the transfer of one or moreanalytes from a biological sample to an array of features on asubstrate, where each feature is associated with a unique spatiallocation on the array. Subsequent analysis of the transferred analytesincludes determining the identity of the analytes and the spatiallocation of the analytes within the biological sample. The spatiallocation of an analyte within the biological sample is determined basedon the feature to which the analyte is bound (e.g., directly orindirectly) on the array, and the feature's relative spatial locationwithin the array.

A “capture probe” refers to any molecule capable of capturing (directlyor indirectly) and/or labelling an analyte (e.g., an analyte ofinterest) in a biological sample. In some embodiments, the capture probeis a nucleic acid or a polypeptide. In some embodiments, the captureprobe includes a barcode (e.g., a spatial barcode and/or a uniquemolecular identifier (UMI)) and a capture domain). In some embodiments,a capture domain can include a sequence that is significantlycomplementary to an analyte, a complement thereof, or a portion thereof(e.g., a capture domain can include a poly-T sequence). In someembodiments, a capture domain can include a sequence that issignificantly complementary to a sequence introduced to the analytebefore capture (e.g., a capture domain can include a sequencecomplementary to a functional domain and/or an adaptor sequence (e.g., atemplate switching oligonucleotide sequence)). In some embodiments, acapture probe can include a cleavage domain and/or a functional domain(e.g., a primer-binding site, such as for next-generation sequencing(NGS)). See, e.g., Section (II)(b) (e.g., subsections (i)-(vi)) of WO2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663.Generation of capture probes can be achieved by any appropriate method,including those described in Section (II)(d)(ii) of WO 2020/176788and/or U.S. Patent Application Publication No. 2020/0277663.

In some embodiments, genetic material is amplified by reversetranscription polymerase chain reaction (RT-PCR). The desired reversetranscriptase activity can be provided by one or more distinct reversetranscriptase enzymes (i.e., RNA dependent DNA polymerases), suitableexamples of which include, but are not limited to: M-MLV, MuLV, AMV,HIV, ArrayScript™, MultiScribe™, ThermoScript™, and SuperScript® I, II,III, and IV enzymes. “Reverse transcriptase” includes not only naturallyoccurring enzymes, but all such modified derivatives thereof, includingalso derivatives of naturally-occurring reverse transcriptase enzymes.

In addition, reverse transcription can be performed usingsequence-modified derivatives or mutants of M-MLV, MuLV, AMV, and HIVreverse transcriptase enzymes, including mutants that retain at leastsome of the functional, e.g., reverse transcriptase, activity of thewild-type sequence. The reverse transcriptase enzyme can be provided aspart of a composition that includes other components, e.g., stabilizingcomponents that enhance or improve the activity of the reversetranscriptase enzyme, such as RNase inhibitor(s), inhibitors ofDNA-dependent DNA synthesis, e.g., actinomycin D. Many sequence-modifiedderivative or mutants of reverse transcriptase enzymes, e.g., M-MLV, andcompositions including unmodified and modified enzymes are commerciallyavailable, e.g., ArrayScript™, MultiScribe™, ThermoScript™, andSuperScript® I, II, III, and IV enzymes.

Certain reverse transcriptase enzymes (e.g., Avian Myeloblastosis Virus(AMV) Reverse Transcriptase and Moloney Murine Leukemia Virus (M-MuLV,MMLV) Reverse Transcriptase) can synthesize a complementary DNA strandusing both RNA (cDNA synthesis) and single-stranded DNA (ssDNA) as atemplate. Thus, in some embodiments, the reverse transcription reactioncan use an enzyme (reverse transcriptase) that is capable of using bothRNA and ssDNA as the template for an extension reaction, e.g., an AMV orMMLV reverse transcriptase.

In some embodiments, the quantification of RNA and/or DNA is carried outby real-time PCR (also known as quantitative PCR or qPCR), usingtechniques well known in the art, such as but not limited to “TAQMAN™”,or dyes such as “SYBR®”, or on capillaries (“LightCycler® Capillaries”).In some embodiments, the quantification of genetic material isdetermined by optical absorbance and with real-time PCR. In someembodiments, the quantification of genetic material is determined bydigital PCR. In some embodiments, the genes analyzed can be compared toa reference nucleic acid extract (DNA and RNA) corresponding to theexpression (mRNA) and quantity (DNA) in order to compare expressionlevels of the target nucleic acids.

A “template switching oligonucleotide” (TSO) is an oligonucleotide thathybridizes to untemplated nucleotides added by a reverse transcriptase(e.g., enzyme with terminal transferase activity) during reversetranscription. In some embodiments, a template switching oligonucleotidehybridizes to untemplated poly(C) nucleotides added by a reversetranscriptase. In some embodiments, the template switchingoligonucleotide adds a common 5′ sequence to full-length cDNA that isused for cDNA amplification.

In some embodiments, the template switching oligonucleotide adds acommon sequence onto the 5′ end of the RNA being reverse transcribed.For example, a template switching oligonucleotide can hybridize tountemplated poly(C) nucleotides added onto the end of a cDNA moleculeand provide a template for the reverse transcriptase to continuereplication to the 5′ end of the template switching oligonucleotide,thereby generating full-length cDNA ready for further amplification. Insome embodiments, once a full-length cDNA molecule is generated, thetemplate switching oligonucleotide can serve as a primer in a cDNAamplification reaction.

In some embodiments, a template switching oligonucleotide is addedbefore, contemporaneously with, or after a reverse transcription, orother terminal transferase-based reaction. In some embodiments, atemplate switching oligonucleotide or complement thereof is included inthe capture probe. In some embodiments, the TSO, or complement thereof,in the capture probe serves as a capture domain. In certain embodiments,methods of sample analysis using template switching oligonucleotides caninvolve the generation of nucleic acid products from analytes of thetissue sample, followed by further processing of the nucleic acidproducts with the template switching oligonucleotide.

Template switching oligonucleotides can include a hybridization regionand a template region. The hybridization region can include any sequencecapable of hybridizing to the target sequence. In some embodiments, thehybridization region can, e.g., include a series of G bases tocomplement the overhanging C bases at the 3′ end of a cDNA molecule. Theseries of G bases can include 1 G base, 2 G bases, 3 G bases, 4 G bases,5 G bases, or more than 5 G bases. The template sequence can include anysequence to be incorporated into the cDNA. In other embodiments, thehybridization region can include at least one base in addition to atleast one G base. In other embodiments, the hybridization can includebases that are not a G base. In some embodiments, the template regionincludes at least 1 (e.g., at least 2, 3, 4, 5 or more) tag sequencesand/or functional sequences. In some embodiments, the template regionand hybridization region are separated by a spacer.

In some embodiments, the template regions include a barcode sequence.The barcode sequence can act as a spatial barcode and/or as a uniquemolecular identifier. In some embodiments, the template region caninclude a functional region, for example a region that can be used foramplification, a region that is complementary to a capture domain on acapture probe, etc. In some embodiments, the template region can includea barcode and/or a unique molecular identifier and/or a functionalsequence and/or a capture domain sequence. Template switchingoligonucleotides can include deoxyribonucleic acids; ribonucleic acids;modified nucleic acids including 2-aminopurine, 2,6-diaminopurine(2-amino-dA), inverted dT, 5-methyl dC, 2′-deoxyInosine, Super T(5-hydroxybutynl-2′-deoxyuridine), Super G (8-aza-7-deazaguanosine),locked nucleic acids (LNAs), unlocked nucleic acids (UNAs, e.g., UNA-A,UNA-U, UNA-C, UNA-G), Iso-dG, Iso-dC, 2′ fluoro bases (e.g., Fluoro C,Fluoro U, Fluoro A, and Fluoro G), or any combination of the foregoing.

In some embodiments, the length of a template switching oligonucleotidecan be at least about 1, 2, 10, 20, 50, 75, 100, 150, 200, or 250nucleotides or longer. In some embodiments, the length of a templateswitching oligonucleotide can be at most about 2, 10, 20, 50, 100, 150,200, or 250 nucleotides or longer.

In some embodiments, more than one analyte type (e.g., nucleic acids andproteins) from a biological sample can be detected (e.g., simultaneouslyor sequentially) using any appropriate multiplexing technique, such asthose described in Section (IV) of WO 2020/176788 and/or U.S. PatentApplication Publication No. 2020/0277663.

In some embodiments, detection of one or more analytes (e.g., proteinanalytes) can be performed using one or more analyte capture agents. Asused herein, an “analyte capture agent” refers to an agent thatinteracts with an analyte (e.g., an analyte in a biological sample) andwith a capture probe (e.g., a capture probe attached to a substrate or afeature) to identify the analyte. In some embodiments, the analytecapture agent includes: (i) an analyte binding moiety (e.g., that bindsto an analyte), for example, an antibody or antigen-binding fragmentthereof; (ii) analyte binding moiety barcode; and (iii) an analytecapture sequence. As used herein, the term “analyte binding moietybarcode” refers to a barcode that is associated with or otherwiseidentifies the analyte binding moiety. As used herein, the term “analytecapture sequence” refers to a region or moiety configured to hybridizeto, bind to, couple to, or otherwise interact with a capture domain of acapture probe. In some cases, an analyte binding moiety barcode (orportion thereof) may be able to be removed (e.g., cleaved) from theanalyte capture agent. Additional description of analyte capture agentscan be found in Section (II)(b)(ix) of WO 2020/176788 and/or Section(II)(b)(viii) U.S. Patent Application Publication No. 2020/0277663.

There are at least two methods to associate a spatial barcode with oneor more neighboring cells, such that the spatial barcode identifies theone or more cells, and/or contents of the one or more cells, asassociated with a particular spatial location. One method is to promoteanalytes or analyte proxies (e.g., intermediate agents) out of a celland towards a spatially-barcoded array (e.g., includingspatially-barcoded capture probes). Another method is to cleavespatially-barcoded capture probes from an array and promote thespatially-barcoded capture probes towards and/or into or onto thebiological sample.

In some cases, capture probes may be configured to prime, replicate, andconsequently yield optionally barcoded extension products from atemplate (e.g., a DNA or RNA template, such as an analyte or anintermediate agent (e.g., a ligation product or an analyte captureagent), or a portion thereof), or derivatives thereof (see, e.g.,Section (II)(b)(vii) of WO 2020/176788 and/or U.S. Patent ApplicationPublication No. 2020/0277663 regarding extended capture probes). In somecases, capture probes may be configured to form ligation products with atemplate (e.g., a DNA or RNA template, such as an analyte or anintermediate agent, or portion thereof), thereby creating ligationproducts that serve as proxies for a template.

As used herein, an “extended capture probe” refers to a capture probehaving additional nucleotides added to the terminus (e.g., 3′ or 5′ end)of the capture probe thereby extending the overall length of the captureprobe. For example, an “extended 3′ end” indicates additionalnucleotides were added to the most 3′ nucleotide of the capture probe toextend the length of the capture probe, for example, by polymerizationreactions used to extend nucleic acid molecules including templatedpolymerization catalyzed by a polymerase (e.g., a DNA polymerase or areverse transcriptase). In some embodiments, extending the capture probeincludes adding to a 3′ end of a capture probe a nucleic acid sequencethat is complementary to a nucleic acid sequence of an analyte orintermediate agent specifically bound to the capture domain of thecapture probe. In some embodiments, the capture probe is extended usingreverse transcription.

In some embodiments, the capture probe is extended using one or more DNApolymerases. The extended capture probes include the sequence of thecapture probe and the sequence of the spatial barcode of the captureprobe.

In some embodiments, extended capture probes are amplified (e.g., inbulk solution or on the array) to yield quantities that are sufficientfor downstream analysis, e.g., via DNA sequencing. In some embodiments,extended capture probes (e.g., DNA molecules) act as templates for anamplification reaction (e.g., a polymerase chain reaction).

Additional variants of spatial analysis methods, including in someembodiments, an imaging step, are described in Section (II)(a) of WO2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663.Analysis of captured analytes (and/or intermediate agents or portionsthereof), for example, including sample removal, extension of captureprobes, sequencing (e.g., of a cleaved extended capture probe and/or acDNA molecule complementary to an extended capture probe), sequencing onthe array (e.g., using, for example, in situ hybridization or in situligation approaches), temporal analysis, and/or proximity capture, isdescribed in Section (II)(g) of WO 2020/176788 and/or U.S. PatentApplication Publication No. 2020/0277663. Some quality control measuresare described in Section (II)(h) of WO 2020/176788 and/or U.S. PatentApplication Publication No. 2020/0277663.

Spatial information can provide information of biological and/or medicalimportance. For example, the methods and compositions described hereincan allow for: identification of one or more biomarkers (e.g.,diagnostic, prognostic, and/or for determination of efficacy of atreatment) of a disease or disorder; identification of a candidate drugtarget for treatment of a disease or disorder; identification (e.g.,diagnosis) of a subject as having a disease or disorder; identificationof stage and/or prognosis of a disease or disorder in a subject;identification of a subject as having an increased likelihood ofdeveloping a disease or disorder; monitoring of progression of a diseaseor disorder in a subject; determination of efficacy of a treatment of adisease or disorder in a subject; identification of a patientsubpopulation for which a treatment is effective for a disease ordisorder; modification of a treatment of a subject with a disease ordisorder; selection of a subject for participation in a clinical trial;and/or selection of a treatment for a subject with a disease ordisorder.

Spatial information can provide information of biological importance.For example, the methods and compositions described herein can allowfor: identification of transcriptome and/or proteome expression profiles(e.g., in healthy and/or diseased tissue); identification of multipleanalyte types in close proximity (e.g., nearest neighbor analysis);determination of up- and/or down-regulated genes and/or proteins indiseased tissue; characterization of tumor microenvironments;characterization of tumor immune responses; characterization of cellstypes and their co-localization in tissue; and identification of geneticvariants within tissues (e.g., based on gene and/or protein expressionprofiles associated with specific disease or disorder biomarkers).

Typically, for spatial array-based methods, a substrate functions as asupport for direct or indirect attachment of capture probes to featuresof the array. A “feature” is an entity that acts as a support orrepository for various molecular entities used in spatial analysis. Insome embodiments, some or all of the features in an array arefunctionalized for analyte capture. Exemplary substrates are describedin Section (II)(c) of WO 2020/176788 and/or U.S. Patent ApplicationPublication No. 2020/0277663. Exemplary features and geometricattributes of an array can be found in Sections (II)(d)(i),(II)(d)(iii), and (II)(d)(iv) of WO 2020/176788 and/or U.S. PatentApplication Publication No. 2020/0277663.

Generally, analytes and/or intermediate agents (or portions thereof) canbe captured when contacting a biological sample with a substrateincluding capture probes (e.g., a substrate with capture probesembedded, spotted, printed, fabricated on the substrate, or a substratewith features (e.g., beads, wells) comprising capture probes). As usedherein, “contact,” “contacted,” and/or “contacting,” a biological samplewith a substrate refers to any contact (e.g., direct or indirect) suchthat capture probes can interact (e.g., bind covalently ornon-covalently (e.g., hybridize)) with analytes from the biologicalsample. Capture can be achieved actively (e.g., using electrophoresis)or passively (e.g., using diffusion). Analyte capture is furtherdescribed in Section (II)(e) of WO 2020/176788 and/or U.S. PatentApplication Publication No. 2020/0277663.

In some cases, spatial analysis can be performed by attaching and/orintroducing a molecule (e.g., a peptide, a lipid, or a nucleic acidmolecule) having a barcode (e.g., a spatial barcode) to a biologicalsample (e.g., to a cell in a biological sample). In some embodiments, aplurality of molecules (e.g., a plurality of nucleic acid molecules)having a plurality of barcodes (e.g., a plurality of spatial barcodes)are introduced to a biological sample (e.g., to a plurality of cells ina biological sample) for use in spatial analysis. In some embodiments,after attaching and/or introducing a molecule having a barcode to abiological sample, the biological sample can be physically separated(e.g., dissociated) into single cells or cell groups for analysis. Somesuch methods of spatial analysis are described in Section (III) of WO2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663.

In some cases, spatial analysis can be performed by detecting multipleoligonucleotides that hybridize to an analyte. In some instances, forexample, spatial analysis can be performed using RNA-templated ligation(RTL). Methods of RTL have been described previously. See, e.g., Credleet al., Nucleic Acids Res. 2017 Aug. 21; 45(14):e128. Typically, RTLincludes hybridization of two oligonucleotides to adjacent sequences onan analyte (e.g., an RNA molecule, such as an mRNA molecule). In someinstances, the oligonucleotides are DNA molecules. In some instances,one of the oligonucleotides includes at least two ribonucleic acid basesat the 3′ end and/or the other oligonucleotide includes a phosphorylatednucleotide at the 5′ end. In some instances, one of the twooligonucleotides includes a capture domain (e.g., a poly(A) sequence, anon-homopolymeric sequence). After hybridization to the analyte, aligase (e.g., SplintR ligase) ligates the two oligonucleotides together,creating a ligation product. In some instances, the two oligonucleotideshybridize to sequences that are not adjacent to one another. Forexample, hybridization of the two oligonucleotides creates a gap betweenthe hybridized oligonucleotides. In some instances, a polymerase (e.g.,a DNA polymerase) can extend one of the oligonucleotides prior toligation. After ligation, the ligation product is released from theanalyte. In some instances, the ligation product is released using anendonuclease (e.g., RNAse H). The released ligation product can then becaptured by capture probes (e.g., instead of direct capture of ananalyte) on an array, optionally amplified, and sequenced, thusdetermining the location and optionally the abundance of the analyte inthe biological sample.

During analysis of spatial information, sequence information for aspatial barcode associated with an analyte is obtained, and the sequenceinformation can be used to provide information about the spatialdistribution of the analyte in the biological sample. Various methodscan be used to obtain the spatial information. In some embodiments,specific capture probes and the analytes they capture are associatedwith specific locations in an array of features on a substrate. Forexample, specific spatial barcodes can be associated with specific arraylocations prior to array fabrication, and the sequences of the spatialbarcodes can be stored (e.g., in a database) along with specific arraylocation information, so that each spatial barcode uniquely maps to aparticular array location.

Alternatively, specific spatial barcodes can be deposited atpredetermined locations in an array of features during fabrication suchthat at each location, only one type of spatial barcode is present sothat spatial barcodes are uniquely associated with a single feature ofthe array. Where necessary, the arrays can be decoded using any of themethods described herein so that spatial barcodes are uniquelyassociated with array feature locations, and this mapping can be storedas described above.

When sequence information is obtained for capture probes and/or analytesduring analysis of spatial information, the locations of the captureprobes and/or analytes can be determined by referring to the storedinformation that uniquely associates each spatial barcode with an arrayfeature location. In this manner, specific capture probes and capturedanalytes are associated with specific locations in the array offeatures. Each array feature location represents a position relative toa coordinate reference point (e.g., an array location, a fiducialmarker) for the array. Accordingly, each feature location has an“address” or location in the coordinate space of the array.

Some exemplary spatial analysis workflows are described in the ExemplaryEmbodiments section of WO 2020/176788 and/or U.S. Patent ApplicationPublication No. 2020/0277663. See, for example, the Exemplary embodimentstarting with “In some non-limiting examples of the workflows describedherein, the sample can be immersed . . . ” of WO 2020/176788 and/or U.S.Patent Application Publication No. 2020/0277663. See also, e.g., theVisium Spatial Gene Expression Reagent Kits User Guide (e.g., Rev C,dated June 2020), and/or the Visium Spatial Tissue Optimization ReagentKits User Guide (e.g., Rev C, dated July 2020).

In some embodiments, spatial analysis can be performed using dedicatedhardware and/or software, such as any of the systems described inSections (II)(e)(ii) and/or (V) of WO 2020/176788 and/or U.S. PatentApplication Publication No. 2020/0277663, or any of one or more of thedevices or methods described in Sections Control Slide for Imaging,Methods of Using Control Slides and Substrates for, Systems of UsingControl Slides and Substrates for Imaging, and/or Sample and ArrayAlignment Devices and Methods, Informational labels of WO 2020/123320.

Suitable systems for performing spatial analysis can include componentssuch as a chamber (e.g., a flow cell or sealable, fluid-tight chamber)for containing a biological sample. The biological sample can be mountedfor example, in a biological sample holder. One or more fluid chamberscan be connected to the chamber and/or the sample holder via fluidconduits, and fluids can be delivered into the chamber and/or sampleholder via fluidic pumps, vacuum sources, or other devices coupled tothe fluid conduits that create a pressure gradient to drive fluid flow.One or more valves can also be connected to fluid conduits to regulatethe flow of reagents from reservoirs to the chamber and/or sampleholder.

The systems can optionally include a control unit that includes one ormore electronic processors, an input interface, an output interface(such as a display), and a storage unit (e.g., a solid state storagemedium such as, but not limited to, a magnetic, optical, or other solidstate, persistent, writeable and/or re-writeable storage medium). Thecontrol unit can optionally be connected to one or more remote devicesvia a network. The control unit (and components thereof) can generallyperform any of the steps and functions described herein. Where thesystem is connected to a remote device, the remote device (or devices)can perform any of the steps or features described herein. The systemscan optionally include one or more detectors (e.g., CCD, CMOS) used tocapture images. The systems can also optionally include one or morelight sources (e.g., LED-based, diode-based, lasers) for illuminating asample, a substrate with features, analytes from a biological samplecaptured on a substrate, and various control and calibration media.

The systems can optionally include software instructions encoded and/orimplemented in one or more of tangible storage media and hardwarecomponents such as application specific integrated circuits. Thesoftware instructions, when executed by a control unit (and inparticular, an electronic processor) or an integrated circuit, can causethe control unit, integrated circuit, or other component executing thesoftware instructions to perform any of the method steps or functionsdescribed herein.

In some cases, the systems described herein can detect (e.g., registeran image) the biological sample on the array. Exemplary methods todetect the biological sample on an array are described in PCTApplication No. 2020/061064 and/or U.S. patent application Ser. No.16/951,854.

Prior to transferring analytes from the biological sample to the arrayof features on the substrate, the biological sample can be aligned withthe array. Alignment of a biological sample and an array of featuresincluding capture probes can facilitate spatial analysis, which can beused to detect differences in analyte presence and/or level withindifferent positions in the biological sample, for example, to generate athree-dimensional map of the analyte presence and/or level. Exemplarymethods to generate a two- and/or three-dimensional map of the analytepresence and/or level are described in PCT Application No. 2020/053655and spatial analysis methods are generally described in WO 2020/061108and/or U.S. patent application Ser. No. 16/951,864.

In some cases, a map of analyte presence and/or level can be aligned toan image of a biological sample using one or more fiducial markers,e.g., objects placed in the field of view of an imaging system whichappear in the image produced, as described in the Substrate AttributesSection, Control Slide for Imaging Section of WO 2020/123320, PCTApplication No. 2020/061066, and/or U.S. patent application Ser. No.16/951,843. Fiducial markers can be used as a point of reference ormeasurement scale for alignment (e.g., to align a sample and an array,to align two substrates, to determine a location of a sample or array ona substrate relative to a fiducial marker) and/or for quantitativemeasurements of sizes and/or distances.

Spatial 5′ Gene Expression Libraries

Provided herein are methods of identifying a location of a targetnucleic acid in a permeabilized biological sample, the methodcomprising: (a) generating a cDNA molecule comprising a sequence that issubstantially complementary to the target nucleic acid using a reversetranscription primer comprising (i) a sequence that is substantiallycomplementary to a portion of the target nucleic acid and (ii) a firstadaptor sequence, wherein the step of generating the cDNA moleculeoccurs within the permeabilized biological sample; (b) ligating a secondadaptor sequence to a 3′ end of the cDNA molecule, wherein the step ofligating is performed within the biological sample; (c) releasing thecDNA molecule from the target nucleic acid, such that the cDNA contactsan array, wherein the array comprises an attached capture probecomprising in a 5′ to a 3′ direction: (i) a spatial barcode and (ii) acapture domain that binds specifically to the second adaptor sequenceligated to the cDNA; (d) extending a 3′ end of the capture probe usingthe cDNA as a template; and (e) determining (i) all or a part of thesequence of the target nucleic acid, or a complement thereof, and (ii)all or a part of the sequence of the spatial barcode, or a complementthereof, and using the determined sequences of (i) and (ii) to identifythe location of the target nucleic acid in the permeabilized biologicalsample.

Also provided herein are methods of identifying a location of a targetnucleic acid in a permeabilized biological sample, the methodcomprising: (a) generating a cDNA molecule comprising a sequence that issubstantially complementary to the target nucleic acid using a reversetranscription primer comprising (i) a sequence that is substantiallycomplementary to a portion of the target nucleic acid and (ii) a firstadaptor sequence, wherein the step of generating the cDNA moleculeoccurs within the permeabilized biological sample; (b) extending a 3′end of the cDNA molecule to include a second adaptor sequence, whereinthe step of extending is performed within the biological sample; (c)releasing the cDNA molecule from the target nucleic acid, such that thecDNA contacts an array, wherein the array comprises an attached captureprobe comprising in a 5′ to a 3′ direction: (i) a spatial barcode and(ii) a capture domain that binds specifically to the second adaptorsequence ligated to the cDNA; (d) extending a 3′ end of the captureprobe using the cDNA as a template; and (e) determining (i) all or apart of the sequence of the target nucleic acid, or a complementthereof, and (ii) all or a part of the sequence of the spatial barcode,or a complement thereof, and using the determined sequences of (i) and(ii) to identify the location of the target nucleic acid in thepermeabilized biological sample. In some such embodiments, the methodcan further comprise hybridizing a template switching oligonucleotide(TSO) to the cDNA molecule. Therefore, in some embodiments, (b)comprises extending a 3′ end of the cDNA molecule to include acomplement of a TSO. In some cases, the TSO can be added to the sampleat the same time as the reverse transcriptase primer.

In some embodiments, steps (a) through (c) are performed when thebiological sample is disposed on the array. In some embodiments, step(a) is performed when the biological sample is not disposed on the arrayand step (b) is performed when the biological sample is disposed on thearray, and wherein the method further comprises between steps (a) and(b), a step of disposing the biological sample on the array. In someembodiments, steps (a) and (b) are performed when the biological sampleis not disposed on the array, and wherein the method further comprisesbetween steps (b) and (c), a step of disposing the biological sample onthe array.

In some embodiments of any of the methods described herein, thebiological sample can be any of the exemplary permeabilized biologicalsamples described herein (e.g., a permeabilized tissue sample, e.g., apermeabilized permeabilized tissue section), or any of the samedescribed in, e.g., Section (I)(d) (e.g., (I)(d)(i) and/or(I)(d)(ii)(13)) of WO 2020/176788 and/or U.S. Patent ApplicationPublication No. 2020/0277663. Some embodiments described herein canoptionally further include a step of permeabilizing the biologicalsample (e.g., using any of the exemplary methods and agents forpermeabilizing a biological sample described herein). In someembodiments, the target nucleic acid is RNA (e.g., mRNA).

In some embodiments, the reverse transcription primer can have a totalof about 10 nucleotides to about 250 nucleotides (e.g., about 10nucleotides to about 225 nucleotides, about 10 nucleotides to about 200nucleotides, about 10 nucleotides to about 175 nucleotides, about 10nucleotides to about 150 nucleotides, about 10 nucleotides to about 125nucleotides, about 10 nucleotides to about 100 nucleotides, about 10nucleotides to about 80 nucleotides, about 10 nucleotides to about 60nucleotides, about 10 nucleotides to about 40 nucleotides, about 10nucleotides to about 20 nucleotides, about 20 nucleotides to about 250nucleotides, about 20 nucleotides to about 225 nucleotides, about 20nucleotides to about 200 nucleotides, about 20 nucleotides to about 175nucleotides, about 20 nucleotides to about 150 nucleotides, about 20nucleotides to about 125 nucleotides, about 20 nucleotides to about 100nucleotides, about 20 nucleotides to about 80 nucleotides, about 20nucleotides to about 60 nucleotides, about 20 nucleotides to about 40nucleotides, about 40 nucleotides to about 250 nucleotides, about 40nucleotides to about 225 nucleotides, about 40 nucleotides to about 200nucleotides, about 40 nucleotides to about 175 nucleotides, about 40nucleotides to about 150 nucleotides, about 40 nucleotides to about 125nucleotides, about 40 nucleotides to about 100 nucleotides, about 40nucleotides to about 80 nucleotides, about 40 nucleotides to about 60nucleotides, about 60 nucleotides to about 250 nucleotides, about 60nucleotides to about 225 nucleotides, about 60 nucleotides to about 200nucleotides, about 60 nucleotides to about 175 nucleotides, about 60nucleotides to about 150 nucleotides, about 60 nucleotides to about 125nucleotides, about 60 nucleotides to about 100 nucleotides, about 60nucleotides to about 80 nucleotides, about 80 nucleotides to about 250nucleotides, about 80 nucleotides to about 225 nucleotides, about 80nucleotides to about 200 nucleotides, about 80 nucleotides to about 175nucleotides, about 80 nucleotides to about 150 nucleotides, about 80nucleotides to about 125 nucleotides, about 80 nucleotides to about 100nucleotides, about 100 nucleotides to about 250 nucleotides, about 100nucleotides to about 225 nucleotides, about 100 nucleotides to about 200nucleotides, about 100 nucleotides to about 175 nucleotides, about 100nucleotides to about 150 nucleotides, about 100 nucleotides to about 125nucleotides, about 125 nucleotides to about 250 nucleotides, about 125nucleotides to about 225 nucleotides, about 125 nucleotides to about 200nucleotides, about 125 nucleotides to about 175 nucleotides, about 125nucleotides to about 150 nucleotides, about 150 nucleotides to about 250nucleotides, about 150 nucleotides to about 225 nucleotides, about 150nucleotides to about 200 nucleotides, about 150 nucleotides to about 175nucleotides, about 175 nucleotides to about 250 nucleotides, about 175nucleotides to about 225 nucleotides, about 175 nucleotides to about 200nucleotides, about 200 nucleotides to about 250 nucleotides, about 200nucleotides to about 225 nucleotides, or about 225 nucleotides to about250 nucleotides).

In some embodiments of any of the methods described herein, the firstadaptor sequence has a total of about 5 nucleotides to about 125nucleotides (e.g., about 5 nucleotides to about 100 nucleotides, about 5nucleotides to about 90 nucleotides, about 5 nucleotides to about 80nucleotides, about 5 nucleotides to about 70 nucleotides, about 5nucleotides to about 60 nucleotides, about 5 nucleotides to about 50nucleotides, about 5 nucleotides to about 45 nucleotides, about 5nucleotides to about 40 nucleotides, about 5 nucleotides to about 35nucleotides, about 5 nucleotides to about 30 nucleotides, about 5nucleotides to about 25 nucleotides, about 5 nucleotides to about 20nucleotides, about 5 nucleotides to about 15 nucleotides, about 5nucleotides to about 10 nucleotides, about 10 nucleotides to about 125nucleotides, about 10 nucleotides to about 100 nucleotides, about 10nucleotides to about 90 nucleotides, about 10 nucleotides to about 80nucleotides, about 10 nucleotides to about 70 nucleotides, about 10nucleotides to about 60 nucleotides, about 10 nucleotides to about 50nucleotides, about 10 nucleotides to about 45 nucleotides, about 10nucleotides to about 40 nucleotides, about 10 nucleotides to about 35nucleotides, about 10 nucleotides to about 30 nucleotides, about 10nucleotides to about 25 nucleotides, about 10 nucleotides to about 20nucleotides, about 10 nucleotides to about 15 nucleotides, about 20nucleotides to about 125 nucleotides, about 20 nucleotides to about 100nucleotides, about 20 nucleotides to about 90 nucleotides, about 20nucleotides to about 80 nucleotides, about 20 nucleotides to about 70nucleotides, about 20 nucleotides to about 60 nucleotides, about 20nucleotides to about 50 nucleotides, about 20 nucleotides to about 45nucleotides, about 20 nucleotides to about 40 nucleotides, about 20nucleotides to about 35 nucleotides, about 20 nucleotides to about 30nucleotides, about 20 nucleotides to about 25 nucleotides, about 30nucleotides to about 125 nucleotides, about 30 nucleotides to about 100nucleotides, about 30 nucleotides to about 90 nucleotides, about 30nucleotides to about 80 nucleotides, about 30 nucleotides to about 70nucleotides, about 30 nucleotides to about 60 nucleotides, about 30nucleotides to about 50 nucleotides, about 30 nucleotides to about 45nucleotides, about 30 nucleotides to about 40 nucleotides, about 30nucleotides to about 35 nucleotides, about 40 nucleotides to about 125nucleotides, about 40 nucleotides to about 100 nucleotides, about 40nucleotides to about 90 nucleotides, about 40 nucleotides to about 80nucleotides, about 40 nucleotides to about 70 nucleotides, about 40nucleotides to about 60 nucleotides, about 40 nucleotides to about 50nucleotides, about 40 nucleotides to about 45 nucleotides, about 50nucleotides to about 125 nucleotides, about 50 nucleotides to about 100nucleotides, about 50 nucleotides to about 90 nucleotides, about 50nucleotides to about 80 nucleotides, about 50 nucleotides to about 70nucleotides, about 50 nucleotides to about 60 nucleotides, about 60nucleotides to about 125 nucleotides, about 60 nucleotides to about 100nucleotides, about 60 nucleotides to about 90 nucleotides, about 60nucleotides to about 80 nucleotides, about 60 nucleotides to about 70nucleotides, about 70 nucleotides to about 125 nucleotides, about 70nucleotides to about 100 nucleotides, about 70 nucleotides to about 90nucleotides, about 70 nucleotides to about 80 nucleotides, about 80nucleotides to about 125 nucleotides, about 80 nucleotides to about 100nucleotides, about 80 nucleotides to about 90 nucleotides, about 90nucleotides to about 125 nucleotides, about 90 nucleotides to about 100nucleotides, or about 100 nucleotides to about 125 nucleotides). In someembodiments, the first adaptor sequence can be any predeterminedsequence. In some embodiments, the first adaptor sequence does notencode a polypeptide and/or can be non-naturally occurring sequence.

In some embodiments the sequence that is substantially complementary(e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least99%, or 100% complementary) to a portion of the sequence of the targetnucleic acid (that is present in the reverse transcription primer) canhave a total of about 10 nucleotides to about 125 nucleotides (or any ofthe subranges of this range described herein). In some embodiments, thesequence that is substantially complementary to a portion of thesequence of the target nucleic acid can be a random sequence. In someembodiments, the sequence that is substantially complementary to aportion of the sequence of the target nucleic acid can include a poly(T)oligonucleotide sequence (e.g., at least 5 contiguous Ts, at least 10continguous Ts, or at least 15 contiguous Ts).

In some embodiments, the step of generating a cDNA molecule including asequence that is substantially complementary to the target nucleic acidusing a reverse transcription primer can include contacting apermeabilized biological sample with a reverse transcriptase (e.g., anyof the exemplary reverse transcriptases described herein or known in theart), dNTPs, and the reverse transcription primer (e.g., any of theexemplary reverse transcription primers described herein). A variety ofkits including a reverse transcriptase and dNTPs are commerciallyavailable. Non-limiting examples of conditions for generating a cDNAmolecule are described herein, and additional examples of conditions forgenerating a cDNA molecule are known in the art.

In some embodiments of any of the methods described herein, the secondadaptor sequence (e.g., ligated to a 3′ end of the generated cDNAmolecule (performed within the biological sample), or included in thegenerated cDNA molecule via extension of a 3′ end of the cDNA molecule)can have a total of about 5 nucleotides to about 125 nucleotides (or anyof the subranges of this range described herein). In some embodiments,the second adaptor sequence can be any predetermined sequence. In someembodiments, the second adaptor sequence does not encode a polypeptideand/or can be non-naturally occurring sequence. In some embodiments, thefirst and second adaptor sequences include different sequences. In someembodiments, the second adaptor sequence can be a template switchingoligonucleotide (TSO) (e.g., any of the exemplary TSOs describedherein), or a complement thereof. In some embodiments, the secondadaptor sequence includes a sequence that is substantially complementary(e.g., at least 80%, at least 85%, at least 90%, at least 92%, at least94%, at least 96%, at least 98%, at least 99%, or 100% complementary) toa sequence in the capture domain of the capture probe.

In some embodiments, the step of ligating the second adaptor sequence toa 3′ end of the generated cDNA molecule (performed within the biologicalsample) can be performed using any of the ligation methods describedherein or known in the art. A wide variety of different methods can beused for ligating nucleic acid molecules, including (but not limited to)“sticky-end” and “blunt-end” ligations. Additionally, single-strandedligation can be used to perform proximity ligation on a single-strandednucleic acid molecule. Sticky-end proximity ligations involve thehybridization of complementary single-stranded sequences between the twonucleic acid molecules to be joined, prior to the ligation event itself.Blunt-end ligations generally do not include hybridization ofcomplementary regions from each nucleic acid molecule because bothnucleic acid molecules lack a single-stranded overhang at the site ofligation. In some embodiments, DNA ligase activity can be provided byone or more distinct DNA ligase enzymes. In some embodiments, the DNAligase enzyme is from a bacterium, e.g., the DNA ligase enzyme is abacterial DNA ligase enzyme. In some embodiments, the DNA ligase enzymeis from a virus (e.g., a bacteriophage). For instance, the DNA ligasecan be T4 DNA ligase. Other enzymes appropriate for the ligation stepinclude, but are not limited to, Tth DNA ligase, Taq DNA ligase,Thermococcus sp. (strain 9oN) DNA ligase (9oN™ DNA ligase, availablefrom New England Biolabs, Ipswich, Mass.), and Ampligase® (availablefrom Lucigen, Middleton, Wis.). Derivatives, e.g., sequence-modifiedderivatives, and/or mutants thereof, can also be used.

For example, the step of ligating can be performed by contacting thepermeabilized biological sample with a ligase and the second adaptorsequence (e.g., a TSO), and optionally, any additional componentsrequired to accelerate the ligation reaction. In some embodiments, themethods can further include blocking the 5′ end of the generated cDNAmolecule prior to the ligating step. Non-limiting examples of conditionsfor performing ligation are described herein, and additional examples ofconditions for performing ligation are known in the art.

In some embodiments, extension of a 3′ end of a generated cDNA toinclude a second adaptor sequence can be performed using any appropriatemethods, such as those described herein for a TSO. For example, the stepof extension of a 3′ end of a generated cDNA molecule can includehybridizing an oligo comprising a complement of the second adaptorsequence to a portion of a 3′ end of the generated cDNA molecule, andextending the cDNA molecule to include the second adaptor sequence. Insome cases, the terminal transferase activity of a reverse transcriptaseenzyme will result in a polymononucleotide sequence (e.g., a poly(C)sequence) at the 3′ end of a generated cDNA molecule, and the oligocomprising a complement of the second adaptor sequence can furtherinclude a complementary polymononucleotide sequence (e.g., a poly(G)sequence) that allows for hybridization to the polymononucleotidesequence of the cDNA molecule. The hybridized oligo comprising thecomplement of the second adaptor sequence can then be used as a templatefor extending the 3′ end of the generated cDNA molecule to include thesecond adaptor sequence. In some cases, the oligo comprising acomplement of the second adaptor sequence is added to the sample at thesame time as the reverse transcription primer.

In some embodiments, the releasing of the cDNA molecule from the targetnucleic acid can be performed by using heat or a chemical denaturant(e.g., KOH).

In some embodiments of any of the methods described herein, the arraycan be any of the types of arrays described herein. For example, thearray includes a slide. In some embodiments, the capture probe isattached to the slide (e.g., by its 5′ end).

In some embodiments, the array is a bead array. In some embodiments, a5′ end of the capture probe is attached to a bead of the bead array.

In some embodiments, the capture probe further comprises a uniquemolecular identifier (UMI) (e.g., a UMI positioned 5′ relative to thecapture domain the capture probe).

In some embodiments, the determining in step (e) comprises sequencing(i) all or a part of the sequence of the target nucleic acid, or acomplement thereof, and (ii) all or a part of the sequence of thespatial barcode, or a complement thereof. In some embodiments, thesequencing is high throughput sequencing, sequencing by hybridization,or any of the other methods for sequencing described herein or known inthe art. For example, sequencing can involve one or more of nucleic acidamplification, the ligation or addition of one or more sequencingadaptors, cleavage of the capture probe from the array, extension of thecapture probe using the bound cDNA as a template, and generating asingle-stranded nucleic acid comprising a sequence that is complementaryto the extended capture probe. Non-limiting methods for determining thesequence of (i) all or a part of the sequence of the target nucleicacid, or a complement thereof, or (ii) all or a part of the sequence ofthe spatial barcode, or a complement thereof, are described herein orare known in the art.

In some embodiments, the methods can optionally further include a stepof imaging the biological sample (e.g., using any of the exemplaryimaging methods described herein or known in the art). In someembodiments, the imaging is performed prior to step (a). In someembodiments, the imaging is performed between steps (b) and (c).

In some embodiments, the method further includes, between steps (b) and(c), a step of freezing and thawing the permeabilized biological sample.In some embodiments, the method can further include, between steps (b)and (c), a step of sectioning (e.g., cryosectioning) the permeabilizedbiological sample.

Exemplary Embodiments

FIG. 1A is an exemplary diagram showing, from left to right, thehybridization of a reverse transcription primer to a target nucleicacid, e.g., an mRNA, within a permeabilized biological sample; thegeneration of a cDNA molecule comprising a sequence that issubstantially complementary to the target nucleic acid, and theaddition/ligation of a second adaptor sequence (e.g., a templateswitching oligonucleotide, or a complement thereof) to the 3′ end of thecDNA molecule within the permeabilized biological sample; and releasingof the cDNA molecule from the target nucleic acid and contacting thereleased cDNA molecule to an array (e.g., any of the exemplary arraysdescribed herein) comprising a capture probe for performance ofadditional steps (e.g., any of the exemplary additional steps describedherein). The second adaptor sequence added (e.g., via extension)/ligatedto the cDNA specifically binds to the capture probe.

FIG. 1B is an exemplary workflow showing the generation of a geneexpression library and the identification of the location of a targetnucleic acid in a biological sample, for example following a targetanalyte capture workflow as shown pictorially in FIG. 1A. Specifically,a biological sample, e.g., a tissue sample, is fixed, stained, andimaged. Any of the exemplary methods described herein or known in theart can be used to fix, stain, and/or image the biological sample. Insome embodiments, the biological sample can be a formalin-fixed andparaffin-embedded (FFPE) tissue sample. In some embodiments, thebiological sample is stained, e.g., using an H&E staining method. Insome embodiments, the tissue sample is fixed, stained, and/or imaged for5 minutes to about 5 hours, e.g., about 5 minutes to about 4.5 hours,about 5 minutes to about 4.0 hours, about 5 minutes to about 3.5 hours,about 5 minutes to about 3.0 hours, about 5 minutes to about 2.5 hours,about 5 minutes to about 2.0 hours, about 5 minutes to about 1.5 hours,about 5 minutes to about 1.0 hour, about 5 minutes to about 50 minutes,about 5 minutes to about 40 minutes, about 5 minutes to about 30minutes, about 5 minutes to about 20 minutes, about 5 minutes to about10 minutes, about 10 minutes to about 5 hours, about 10 minutes to about4.5 hours, about 10 minutes to about 4.0 hours, about 10 minutes toabout 3.5 hours, about 10 minutes to about 3.0 hours, about 10 minutesto about 2.5 hours, about 10 minutes to about 2.0 hours, about 10minutes to about 1.5 hours, about 10 minutes to about 1.0 hour, about 10minutes to about 50 minutes, about 10 minutes to about 40 minutes, about10 minutes to about 30 minutes, about 10 minutes to about 20 minutes,about 20 minutes to about 5 hours, about 20 minutes to about 4.5 hours,about 20 minutes to about 4.0 hours, about 20 minutes to about 3.5hours, about 20 minutes to about 3.0 hours, about 20 minutes to about2.5 hours, about 20 minutes to about 2.0 hours, about 20 minutes toabout 1.5 hours, about 20 minutes to about 1.0 hour, about 20 minutes toabout 50 minutes, about 20 minutes to about 40 minutes, about 20 minutesto about 30 minutes, about 30 minutes to about 5 hours, about 30 minutesto about 4.5 hours, about 30 minutes to about 4.0 hours, about 30minutes to about 3.5 hours, about 30 minutes to about 3.0 hours, about30 minutes to about 2.5 hours, about 30 minutes to about 2.0 hours,about 30 minutes to about 1.5 hours, about 30 minutes to about 1.0 hour,about 30 minutes to about 50 minutes, about 30 minutes to about 40minutes, about 1.0 hour to about 5 hours, about 1.0 hour to about 4.5hours, about 1.0 hour to about 4.0 hours, about 1.0 hour to about 3.5hours, about 1.0 hour to about 3.0 hours, about 1.0 hour to about 2.5hours, about 1.0 hour to about 2.0 hours, about 1.0 hour to about 1.5hours, about 1.5 hour to about 5 hours, about 1.5 hour to about 4.5hours, about 1.5 hour to about 4.0 hours, about 1.5 hour to about 3.5hours, about 1.5 hour to about 3.0 hours, about 1.5 hour to about 2.5hours, about 1.5 hour to about 2.0 hours, about 2.0 hour to about 5hours, about 2.0 hour to about 4.5 hours, about 2.0 hour to about 4.0hours, about 2.0 hour to about 3.5 hours, about 2.0 hour to about 3.0hours, about 2.0 hour to about 2.5 hours, about 2.5 hour to about 5hours, about 2.5 hour to about 4.5 hours, about 2.5 hour to about 4.0hours, about 2.5 hour to about 3.5 hours, about 2.5 hour to about 3.0hours, about 3.0 hour to about 5 hours, about 3.0 hour to about 4.5hours, about 3.0 hour to about 4.0 hours, about 3.0 hour to about 3.5hours, about 3.5 hour to about 5 hours, about 3.5 hour to about 4.5hours, about 3.5 hour to about 4.0 hours, about 4.0 hour to about 5hours, about 4.0 hour to about 4.5 hours, or about 4.5 hour to about 5hours.

After the fixation, staining and imaging of the biological sample, thebiological sample is permeabilized. Permeabilization of the biologicalsample (e.g., a tissue sample) can be performed using any of theexemplary methods or exemplary reagents described herein, or in, e.g.,Section (I)(d)(ii)(13) of WO 2020/176788 and/or U.S. Patent ApplicationPublication No. 2020/0277663. In some embodiments, the permeabilizationof the biological sample (e.g., tissue sample) can be performed forabout 1 minute to about 5 hours (e.g., about 1 minute to about 4.5hours, about 1 minute to about 4.0 hours, about 1 minute to about 3.5hours, about 1 minute to about 3.0 hours, about 1 minute to about 2.5hours, about 1 minute to about 2.0 hours, about 1 minute to about 1.5hours, about 1 minute to about 1.0 hour, about 1 minute to about 50minutes, about 1 minute to about 40 minutes, about 1 minute to about 30minutes, about 1 minute to about 20 minutes, about 1 minute to about 10minutes, about 1 minute to about 5 minutes, about 10 minutes to about5.0 hours, about 10 minutes to about 4.5 hours, about 10 minutes toabout 4.0 hours, about 10 minutes to about 3.5 hours, about 10 minutesto about 3.0 hours, about 10 minutes to about 2.5 hours, about 10minutes to about 2.0 hours, about 10 minutes to about 1.5 hours, about10 minutes to about 1.0 hour, about 10 minutes to about 50 minutes,about 10 minutes to about 40 minutes, about 10 minutes to about 30minutes, about 10 minutes to about 20 minutes, about 20 minutes to about5.0 hours, about 20 minutes to about 4.5 hours, about 20 minutes toabout 4.0 hours, about 20 minutes to about 3.5 hours, about 20 minutesto about 3.0 hours, about 20 minutes to about 2.5 hours, about 20minutes to about 2.0 hours, about 20 minutes to about 1.5 hours, about20 minutes to about 1.0 hour, about 20 minutes to about 50 minutes,about 20 minutes to about 40 minutes, about 20 minutes to about 30minutes, about 30 minutes to about 5.0 hours, about 30 minutes to about4.5 hours, about 30 minutes to about 4.0 hours, about 30 minutes toabout 3.5 hours, about 30 minutes to about 3.0 hours, about 30 minutesto about 2.5 hours, about 30 minutes to about 2.0 hours, about 30minutes to about 1.5 hours, about 30 minutes to about 1.0 hour, about 30minutes to about 50 minutes, about 30 minutes to about 40 minutes, about1.0 hour to about 5.0 hours, about 1.0 hour to about 4.5 hours, about1.0 hour to about 4.0 hours, about 1.0 hour to about 3.5 hours, about1.0 hour to about 3.0 hours, about 1.0 hour to about 2.5 hours, about1.0 hour to about 2.0 hours, about 1.0 hour to about 1.5 hours, about2.0 hours to about 5.0 hours, about 2.0 hours to about 4.5 hours, about2.0 hours to about 4.0 hours, about 2.0 hours to about 3.5 hours, about2.0 hours to about 3.0 hours, about 2.0 hours to about 2.5 hours, about3.0 hours to about 5.0 hours, about 3.0 hours to about 4.5 hours, about3.0 hours to about 4.0 hours, about 3.0 hours to about 3.5 hours, about4.0 hours to about 5.0 hours, about 4.0 hours to about 4.5 hours, orabout 4.5 hours to about 5.0 hours).

After permeabilization of the biological sample, the target nucleic acidin the permeabilized biological sample is contacted and hybridized witha reverse transcription primer (e.g., any of the reverse transcriptionprimers described herein) to generate a cDNA molecule comprising asequence that is substantially complementary to the target nucleic acid.

In some embodiments, after the generation of the cDNA molecule, a secondadaptor sequence is ligated to the 3′ end of the cDNA molecule. Anysuitable adaptor sequence described herein can be ligated to the 3′ endof the cDNA molecule. In some embodiments, the second adaptor moleculeis a template switching oligonucleotide (TSO).

In some embodiments, a 3′ end of the cDNA molecule is extended toinclude a second adaptor sequence. Any adaptor sequence described hereincan be included to the 3′ end of the cDNA molecule. In some embodiments,the second adaptor molecule is a template switching oligonucleotide(TSO), or a complement thereof.

The generation of the cDNA molecule and the addition (e.g., viaextension) or ligation of the second adaptor sequence can be performedover about 1 minute to about 2 hours (e.g., about 1 minute to about 1.5hours, about 1 minute to about 1.0 hour, about 1 minute to about 40minutes, about 1 minute to about 20 minutes, about 1 minute to about 10minutes, about 10 minutes to about 2.0 hours, about 10 minutes to about1.5 hours, about 10 minutes to about 1.0 hour, about 10 minutes to about40 minutes, about 10 minutes to about 20 minutes, about 20 minutes toabout 2.0 hours, about 20 minutes to about 1.5 hours, about 20 minutesto about 1.0 hour, about 20 minutes to about 40 minutes, about 40minutes to about 2.0 hours, about 40 minutes to about 1.5 hours, about40 minutes to about 1.0 hour, about 1.0 hour to about 2.0 hours, about 1hour to about 1.5 hours, or about 1.5 hours to about 2.0 hours).

In some embodiments, the reverse transcription occurs within thepermeabilized biological sample, e.g., a permeabilized tissue sample. Insome embodiments, the permeabilization and the generation of the cDNAmolecule occurs while the biological sample is disposed on the array.

After the generation of the cDNA molecule and the addition (e.g., viaextension) or ligation of the second adaptor sequence, the cDNA moleculecan be denatured/released from the target nucleic acid and the cDNAmolecule is contacted with an array comprising a capture probe. Thecapture probe can include in a 5′ to a 3′ direction: (i) a spatialbarcode and (ii) a capture domain that binds specifically to the secondadaptor sequence ligated to the cDNA. When contacting with the arraywith the cDNA molecule, the attached capture probe captures the cDNAmolecule using the capture domain, and the 3′ end of the capture probeis extended to add a sequence that is substantially complementary to thecDNA molecule sequence. In some embodiments, the methods can furtherinclude generating a single-stranded nucleic acid that includes asequence that is substantially complementary to the extended captureprobe. The optional steps of extending a 3′ end of the capture probe(using the specifically bound cDNA as a template) and generating asingle-stranded nucleic acid including a sequence complementary to theextended capture probe can be performed for about 5 minutes to about 2hours (or any of the subranges within this range described herein).

In some embodiments, a single-stranded nucleic acid including a sequencecomplementary to the extended capture probe can be denatured from theextended capture probe, and optionally, transferred to a different tubeor container for performance of additional steps.

The single-stranded nucleic acid including a sequence complementary tothe extended capture probe can be quantitated and/or sequenced or atleast partially sequenced using any of the methods described herein, ordescribed in, e.g., Sections (II)(a) or (II)(g) (e.g., (II)(g)(iv)) ofWO 2020/176788 and/or U.S. Patent Application Publication No.2020/0277663 or known in the art. In some embodiments, the quantitationand/or sequencing of the single-stranded nucleic acid including asequence complementary to the extended capture probe can be quantitatedand/or sequenced or at least partially sequenced for about 10 minutes toabout 2 hours (or any of the subranges of this range described herein).

Following the denaturing of the single-stranded nucleic acid including asequence complementary to the extended capture probe, thesingle-stranded nucleic acid including the sequence complementary to theextended capture probe can be subjected to amplification, fragmentation,end-repairing, A-tailing, adaptor ligation, sample index PCR, and theconstruction and quality control of a gene expression library, using anyof the exemplary methods described herein, or described in, e.g.,Sections (II)(a) or (II)(g) (e.g., (II)(g)(iv)) of WO 2020/176788 and/orU.S. Patent Application Publication No. 2020/0277663.

FIG. 2A is an exemplary diagram showing, from left to right, thehybridization of a reverse transcription primer to a target nucleicacid, e.g., an mRNA, within a permeabilized biological sample, e.g., awhole tissue sample that has not been sectioned, cut or furtherfragmented; the generation of a cDNA molecule comprising a sequence thatis substantially complementary to the target nucleic acid, and theaddition (e.g., via extension) or ligation of a second adaptor sequence(e.g., a template switching oligonucleotide, or a complement thereof) tothe 3′ end of the cDNA molecule within the permeabilized biologicalsample, e.g., a whole tissue sample that has not been sectioned, cut orfurther fragmented; the fixation and/or flash-freezing of the biologicalsample, and the cryosectioning of the whole tissue sample for use inadditional steps. When using a whole tissue sample that has not beensectioned, cut or further fragmented, the steps described in FIG. 2A areperformed when the biological sample is not disposed on an array.

FIG. 2B is an exemplary workflow showing the generation of a geneexpression library and the identification of the location of a targetnucleic acid in a biological sample, for example following the targetanalyte capture workflow of FIG. 2A. Specifically, a biological sample,e.g., a whole tissue sample, is permeabilized. Any suitablepermeabilization method described herein, or in, e.g., Section(I)(d)(ii)(13) of WO 2020/176788 and/or U.S. Patent ApplicationPublication No. 2020/0277663, or known in the art can be used topermeabilize the whole tissue sample. In some embodiments, thebiological sample is permeabilized for about 5 minutes to about 5 hours(or any of the subranges of this range described herein).

After permeabilization of the biological sample, the target nucleic acidin the permeabilized biological sample is contacted and annealed with areverse transcription primer to generate a cDNA molecule comprising asequence that is substantially complementary to the target nucleic acid.After synthesis of the cDNA molecule, a second adaptor sequence is added(e.g., via extension) or ligated to the 3′ end of the cDNA molecule. Anysuitable adaptor sequence described in the current application can beused to ligate to the 3′ cDNA molecule. In some embodiments, the secondadaptor molecule is a template switching oligonucleotide (TSO), or acomplement thereof. The generation of the cDNA molecule and the addition(e.g., via extension) or ligation of the second adaptor sequence can beperformed for about 10 minutes to about 2 hours (or any of the subrangesof this range described herein). In some embodiments, the synthesis ofthe cDNA molecule occurs within a permeabilized whole tissue sample.

Following the generation of the cDNA molecule, the biological sample,e.g., the whole tissue sample, can be fixed and/or flash-frozen. Anysuitable methods described herein, or in, e.g., Section(I)(d)(1)-(I)(d)(4) of WO 2020/176788 and/or U.S. Patent ApplicationPublication No. 2020/0277663, or known in the art can be used to fix andflash-freeze the tissue sample. In some embodiments, the biologicalsample, e.g., the whole tissue sample is formalin-fixed andparaffin-embedded (FFPE). In some embodiments, the biological sample,e.g., whole tissue sample, is flash-frozen using liquid nitrogen. Theflash-frozen tissue sample is then sectioned for future steps. In someembodiments, the sectioning is performed using cryosectioning. In someembodiments, the methods further comprise a thawing step, after thecryosectioning.

After sectioning, the biological sample, e.g., tissue sample, can bestained, and imaged. Any of the methods described herein, or in, e.g.,Section (I)(d)(6) or (II)(a)(i) of WO 2020/176788 and/or U.S. PatentApplication Publication No. 2020/0277663, or known in the art can beused to stain and/or image the biological sample. In some embodiments,the biological sample is stained using an H&E staining method. In someembodiments, the tissue sample is stained and imaged for about 10minutes to about 2 hours (or any of the subranges of this rangedescribed herein). Additional time may be needed for staining andimaging of different types of biological samples.

In some embodiments, the generation of the cDNA occurs within thepermeabilized biological sample, e.g., a permeabilized tissue sample. Insome embodiments, the permeabilization and the generation of the cDNAoccurs while the biological sample is not disposed on the array.

After the generation of the cDNA molecule, the addition (e.g., viaextension) or ligation of the second adaptor sequence to a 3′ end of thecDNA, and the tissue fixation, freezing, sectioning, staining, andimaging, the cDNA is released/denatured from the target nucleic acid andthe cDNA is contacted with an array comprising a capture probe. Thecapture probe can include in a 5′ to a 3′ direction: (i) a spatialbarcode and (ii) a capture domain that binds specifically to the secondadaptor sequence ligated to the cDNA. When the array is contacted withthe cDNA, the capture probe binds specifically to the cDNA via thecapture domain, and the 3′ end of the capture probe is extended (usingthe specifically bound cDNA as a template) to add a sequence that issubstantially complementary to the cDNA. The method can further includegenerating a single-stranded nucleic acid that is complementary to theextended capture probe. The denaturing of the cDNA from the targetnucleic acid, the extension of the capture probe (using the specificallybound cDNA as a template), and optional generation of a single-strandednucleic acid including a sequence that is complementary to the extendedcapture probe can be performed over 10 minutes to about 5 hours (or anyof the subranges of this range described herein).

The single-stranded nucleic acid including a sequence that iscomplementary to the extended capture probe can be separated/denaturedfrom the extended capture probe and optionally, transferred to acontainer (e.g., a strip tube) for the performance of additional steps.

The extended capture probe and/or a denatured/separated single-strandednucleic acid including a sequence that is complementary to the extendedcapture probe can be quantitated and/or sequenced or at least partiallysequenced using any of the methods described herein, or described in,e.g., Sections (II)(a) or (II)(g) (e.g., (II)(g)(iv)) of WO 2020/176788and/or U.S. Patent Application Publication No. 2020/0277663 or known inthe art. In some embodiments, the quantitation and/or sequencing of theextended capture probe and/or a denatured/separated single-strandednucleic acid including a sequence that is complementary to the extendedcapture probe can be performed for about 10 minutes to about 5 hours (orany of the subranges of this range described herein).

Following the denaturing of the single-stranded nucleic acid including asequence complementary to the extended capture probe, thesingle-stranded nucleic acid including the sequence complementary to theextended capture probe can be subjected to amplification, fragmentation,end-repairing, A-tailing, adaptor ligation, sample index PCR, and theconstruction and quality control of a gene expression library, using anyof the exemplary methods described herein, or described in, e.g.,Sections (II)(a) or (II)(g) (e.g., (II)(g)(iv)) of WO 2020/176788 and/orU.S. Patent Application Publication No. 2020/0277663.

FIG. 3A is a diagram showing an exemplary reaction mix that can be usedto generate a cDNA and that can be used to add (e.g., via extension) orligate a second adaptor sequence to a 3′ end of the cDNA. For example, atarget nucleic acid in the biological sample can be an mRNA moleculehaving a poly(A) tail at the 3′ end of the sequence. For the generationof a cDNA, a reverse transcription primer is added to the biologicalsample. The reverse transcription primer includes, from the 5′ end tothe 3′ end, a first adaptor sequence (SM), and a sequence that issubstantially complementary to a portion of the mRNA. In someembodiments, the sequence that is substantially complementary to aportion of the mRNA includes a poly(T) sequence comprising a sequence ofT_(n), wherein n can be 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30. In someembodiments, the sequence substantially complementary to a portion ofthe mRNA is a dT30VN sequence, where the sequence comprises T_(n),wherein n is 30, wherein V is A, G, or C, and where N is A, G, C, or T.In some embodiments, the sequence substantially complementary to aportion of the mRNA can includes a random sequence.

In some embodiments, the complement of the second adaptor sequence canbe added to a reverse transcription mix. In some embodiments, thecomplement of the second adaptor sequence can be a template switchingoligonucleotide (TSO) having a rGrGrG sequence at the 3′ end of thesecond adaptor sequence.

The reverse transcription step can be performed using a method thatincludes a pre-equilibration thermocycling protocol (e.g., lidtemperature and pre-equilibration at about 53° C., reverse transcriptionat about 53° C. for about 60 minutes, about 90° C. for about 5 min, andthen held at about 4° C.). Any suitable reverse transcriptase andbuffers can be used to perform reverse transcription, such as any ofthose described herein or known in the art. In some embodiments, thereverse transcription mix can further include other components thatassist or increase the rate of a reverse transcription reaction. Forexample, the reverse transcription mix can further include dNTPs. Thethermocycling protocol for the reverse transcription reaction and theligation of the second adaptor sequence to the 3′ end of the cDNA can befurther optimized according to different target nucleic acids indifferent types of biological samples.

The reaction described in FIG. 3A generates a cDNA comprising, for the5′ end to the 3′ end, a first adaptor sequence (e.g., SM sequence), asequence substantially complementary to the target nucleic acid (e.g., adT30VN sequence), a sequence complementary to the target nucleic acidsequence, and a complement of a second adaptor sequence (e.g., a TSOsequence). In some embodiments, the cDNA is hybridized to the targetnucleic acid molecule is subsequently denatured/separated from thenucleic acid analyte.

FIG. 3B is a diagram showing an exemplary array comprising a secondexemplary capture probe. The capture probe comprises, from the 5′ end tothe 3′ end, a linker sequence, a partial R1 primer sequence, a spatialbarcode, a unique molecular identifier (UMI), a capture domain, e.g., asequence substantially complementary to the second adaptor sequence. Insome embodiments the sequence substantially complementary to the secondadaptor sequence is substantially complementary to a template switchingoligonucleotide (TSO) ligated to the cDNA molecule. In some embodimentsthe sequence substantially complementary to the second adaptor sequenceis comprises a template switching oligonucleotide (TSO) used as atemplate for extension of a 3′ end of the cDNA molecule. In someembodiments, the 5′ end of the capture probe is attached to the array.After the capture domain on the capture probe specifically binds to thesecond adaptor sequence on the cDNA molecule, a 3′ end of the captureprobe is extended (using the specifically bound cDNA as a template) toadd a sequence that is substantially complementary to the sequence ofthe cDNA and a sequence complementary to the first adaptor sequence). Inaddition, a single-stranded nucleic acid that includes a sequence thatis complementary to the extended capture probe can be generated (bottomstrand shown in bottom half of figure).

The generation of the extended capture probe and the generation of thesingle-stranded nucleic acid that includes a sequence complementary tothe extended capture probe in FIG. 3B can be performed using athermocycling protocol (e.g., lid temperature and pre-equilibrate atabout 95° C., denaturing at about 95° C. for about 1 min, reannealing atabout 60° C. for about 60 min, extension at about 90° C. for about 5minutes, and then held at about 4° C.). The reaction mixture furtherincludes all necessary polymerase and buffers. In some embodiments, thepolymerase can be a DNA polymerase. In some embodiments, the DNApolymerase can be HotStart Taq DNA polymerase.

After the generation of the single-stranded nucleic acid including asequence that is complementary to the extended capture probe, KOH can beadded to denature the single-stranded nucleic acid including a sequencecomplementary to the extended capture probe from the extended captureprobe, and transferring the single-stranded nucleic acid including asequence that is complementary to the extended capture probe to adifferent tube (e.g., one or more tubes, for example a strip tube thatmight be used in a thermocyling instrument) for the performance ofadditional steps.

FIG. 3C is a diagram showing exemplary steps of amplification,quantitation, and/or sequencing of a single-stranded nucleic acid thatincludes a sequence complementary to the extended capture probe. In someembodiments, the methods can include the performance of qPCR. Exemplarymethods for performing qPCR are described herein and are known in theart.

In some embodiments, the method can result in the generation of asingle-stranded nucleic acid that includes in a 5′ to a 3′ direction, alinker, a partial R1 primer sequence, a spatial barcode, a UMI, asequence complementary to the second adaptor sequence, a sequencepresent in the target nucleic acid, and a sequence complementary to thefirst adaptor sequence.

In some embodiments, the method can result in the generation of asingle-stranded nucleic acid that includes in a 5′ to a 3′ direction, aP5 sequencing handle, a i5 sequencing handle, a linker, a partial R1 orR1 primer sequence, a spatial barcode, a UMI, a sequence complementaryto the second adaptor sequence, a sequence present in the target nucleicacid, a R2 adaptor sequence, an i7 sequencing handle, and a P7sequencing handle.

In some embodiments, the method can result in the generation of asingle-stranded nucleic acid that includes in a 3′ to a 5′ direction, asequence complementary to a linker, a sequence complementary to apartial R1 primer sequence, a sequence complementary to a spatialbarcode, a sequence complementary to a UMI, the second adaptor sequence,a sequence complementary to a sequence present in the target nucleicacid, and the first adaptor sequence.

In some embodiments, the method can result in the generation of asingle-stranded nucleic acid that includes in a 3′ to a 5′ direction, asequence complementary to a P5 sequencing handle, a sequencecomplementary to an i5 sequencing handle, a sequence complementary to alinker, a sequence complementary to a partial R1 or R1 primer sequence,a sequence complementary to a spatial barcode, a sequence complementaryto a UMI, the second adaptor sequence, a sequence complementary to asequence present in the target nucleic acid, a sequence complementary toan R2 adaptor sequence, a sequence complementary to an i7 sequencinghandle, and a sequence complementary to a P7 sequencing handle.

In some embodiments of any of the methods described herein, step (e)includes sequencing all or a part of the sequence of the spatialbarcode, or a complement thereof, and sequencing all of a part of thesequence of the target nucleic acid, or a complement thereof. Thesequencing can be performed using any of the methods described herein.In some embodiments, step (e) includes sequencing the full-lengthsequence of the spatial barcode, or a complement thereof. In someembodiments, step (e) includes sequencing a part of the sequence of thespatial barcode, or a complement thereof. In some embodiments, step (e)includes sequencing the full-length sequence of the target nucleic acid,or a complement thereof. In some embodiments, step (e) includessequencing a part of the target nucleic acid, or a complement thereof.In some embodiments, the sequencing is performed using high throughputsequencing. In some embodiments, the target nucleic acid is sequencedfrom the 5′ end of the target nucleic acid. In some embodiments, thetarget nucleic acid is sequenced from the 3′ end of the target nucleicacid. In some embodiments, the target nucleic acid is sequenced fromboth the 3′ end and the 5′ end of the target nucleic acid. The librarycan be sequenced using available sequencing platforms, including, any ofMiSeq, NextSeq 500/550, HiSeq 2500, HiSeq 3000/4000, NovaSeq, or iSeq.

Kits

Also provided herein are kits for performing any of the methodsdescribed herein. For example, provided herein is a kit comprising: areverse transcription primer comprising (i) a sequence that issubstantially complementary to a portion of the target nucleic acid and(ii) a first adaptor sequence; a reverse transcriptase; and anoligonucleotide comprising a second adaptor sequence or a complementthereof. In some embodiments, the reverse transcriptase is a reversetranscriptase with terminal transferase activity. In some embodiments,the second adaptor sequence or complement thereof is a TSO or complementthereof. The kits can include any other buffers, enzymes, cofactors, orother components useful in the method. For example, when the methodincludes ligating the second adaptor sequence to the generated cDNAmolecule, the kit can further include a ligase. In some embodiments, thekits can also include an array, wherein the array comprises an attachedcapture probe comprising in a 5′ to a 3′ direction: (i) a spatialbarcode and (ii) a capture domain that binds specifically to the secondadaptor sequence. In some examples, the kit can further include apermeabilizing agent. In some embodiments, the kit can further include alipase, a protease, and/or an RNAse.

Exemplary Embodiments

Embodiment 1 is a method of identifying a location of a target nucleicacid in a permeabilized biological sample, the method comprising:

(a) generating a cDNA molecule comprising a sequence that issubstantially complementary to the target nucleic acid using a reversetranscription primer comprising (i) a sequence that is substantiallycomplementary to a portion of the target nucleic acid and (ii) a firstadaptor sequence, wherein the step of generating the cDNA moleculeoccurs within the permeabilized biological sample;

(b) ligating a second adaptor sequence to a 3′ end of the cDNA molecule,wherein the step of ligating is performed within the biological sample;

(c) releasing the cDNA molecule from the target nucleic acid, such thatthe cDNA contacts an array, wherein the array comprises an attachedcapture probe comprising in a 5′ to a 3′ direction: (i) a spatialbarcode and (ii) a capture domain that binds specifically to the secondadaptor sequence ligated to the cDNA;

(d) extending a 3′ end of the capture probe using the cDNA as atemplate; and

(e) determining (i) all or a part of the sequence of the target nucleicacid, or a complement thereof, and (ii) all or a part of the sequence ofthe spatial barcode, or a complement thereof, and using the determinedsequences of (i) and (ii) to identify the location of the target nucleicacid in the permeabilized biological sample.

Embodiment 2 is a method of identifying a location of a target nucleicacid in a permeabilized biological sample, the method comprising:

(a) generating a cDNA molecule comprising a sequence that issubstantially complementary to the target nucleic acid using a reversetranscription primer comprising (i) a sequence that is substantiallycomplementary to a portion of the target nucleic acid and (ii) a firstadaptor sequence, wherein the step of generating the cDNA moleculeoccurs within the permeabilized biological sample;

(b) extending a 3′ end of the cDNA molecule to include a second adaptorsequence, wherein the step of extending is performed within thebiological sample;

(c) releasing the cDNA molecule from the target nucleic acid, such thatthe cDNA contacts an array, wherein the array comprises an attachedcapture probe comprising in a 5′ to a 3′ direction: (i) a spatialbarcode and (ii) a capture domain that binds specifically to the secondadaptor sequence;

(d) extending a 3′ end of the capture probe using the cDNA as atemplate; and

(e) determining (i) all or a part of the sequence of the target nucleicacid, or a complement thereof, and (ii) the sequence of the spatialbarcode, or a complement thereof, and using the determined sequences of(i) and (ii) to identify the location of the target nucleic acid in thepermeabilized biological sample.

Embodiment 3 is the method of Embodiment 2, wherein step (b) occurssimultaneously with step (a).

Embodiment 4 is the method of any one of Embodiments 1-3, wherein steps(a) through (c) are performed when the biological sample is disposed onthe array.

Embodiment 5 is the method of any one of Embodiments 1-3, wherein step(a) is performed when the biological sample is not disposed on the arrayand step (b) is performed when the biological sample is disposed on thearray, and wherein the method further comprises between steps (a) and(b), a step of disposing the biological sample on the array.

Embodiment 6 is the method of any one of Embodiments 1-3, wherein steps(a) and (b) are performed when the biological sample is not disposed onthe array, and wherein the method further comprises between steps (b)and (c), a step of disposing the biological sample on the array.

Embodiment 7 is the method of any one of Embodiments 1-6, wherein thesequence that is substantially complementary to a portion of the targetnucleic acid present in the reverse transcription primer comprises apoly(T) sequence.

Embodiment 8 is the method of any one of Embodiments 1-6, wherein thesequence that is substantially complementary to a portion of the targetnucleic acid present in the reverse transcription primer comprises arandom sequence.

Embodiment 9 is the method of any one of Embodiments 1-8, wherein thesecond adaptor sequence is a template switching oligonucleotide (TSO),or a complement thereof.

Embodiment 10 is the method of any one of Embodiments 1-9, wherein thearray comprises a slide.

Embodiment 11 is the method of Embodiment 10, wherein a 5′ end of thecapture probe is attached to the slide.

Embodiment 12 is the method of any one of Embodiments 1-9, wherein thearray is a bead array.

Embodiment 13 is the method of Embodiment 12, wherein a 5′ end of thecapture probe is attached to a bead of the bead array.

Embodiment 14 is the method of any one of Embodiments 1-13, wherein thecapture probe further comprises a unique molecular identifier (UMI).

Embodiment 15 is the method of Embodiment 14, wherein the UMI ispositioned 5′ relative to the capture domain in the capture probe.

Embodiment 16 is the method of any one of Embodiments 1-15, wherein thedetermining in step (e) comprises sequencing (i) all or a part of thesequence of the target nucleic acid, or a complement thereof, and (ii)the sequence of the spatial barcode, or a complement thereof.

Embodiment 17 is the method of Embodiment 16, wherein the sequencing ishigh throughput sequencing.

Embodiment 18 is the method of Embodiment 16, wherein the sequencing issequencing by hybridization.

Embodiment 19 is the method of any one of Embodiments 1-18, wherein thetarget nucleic acid is RNA.

Embodiment 20 is the method of Embodiment 19, wherein the RNA is anmRNA.

Embodiment 21 is the method of any one of Embodiments 1-20, wherein thepermeabilized biological sample is a permeabilized tissue section.

Embodiment 22 is the method of Embodiment 21, wherein the permeabilizedtissue section is a permeabilized formalin-fixed and paraffin-embedded(FFPE) tissue section.

Embodiment 23 is the method of any one of Embodiments 1-22, wherein themethod further comprises a step of imaging the biological sample.

Embodiment 24 is the method of Embodiment 23, wherein the step ofimaging is performed prior to step (a).

Embodiment 25 is the method of Embodiment 24, wherein the step ofimaging is performed between steps (b) and (c).

Embodiment 26 is the method of any one of Embodiments 1-3 and 6-25,wherein the method further comprises, between steps (b) and (c), a stepof freezing and thawing the permeabilized biological sample.

Embodiment 27 is the method of Embodiment 26, wherein the method furthercomprises, between steps (b) and (c), a step of sectioning thepermeabilized biological sample.

Embodiment 28 is the method of Embodiment 27, wherein the step ofsectioning the permeabilized biological sample is performed usingcryosectioning.

Embodiment 29 is the method of any one of Embodiments 1-28, wherein themethod further comprises, prior to step (a), a step of permeabilizingthe biological sample.

Embodiment 30 is the method of any one of Embodiments 1-29, wherein theperformance of step (a) comprises introducing a reverse transcriptase,dNTPs, and the reverse transcription primer into the permeabilizedbiological sample.

Embodiment 31 is a kit comprising: a reverse transcription primercomprising (i) a sequence that is substantially complementary to aportion of the target nucleic acid and (ii) a first adaptor sequence; areverse transcriptase; and an oligonucleotide comprising a secondadaptor sequence or a complement thereof.

Embodiment 32 is the kit of Embodiment 31, wherein the kit furthercomprises a ligase.

Embodiment 33 is the kit of Embodiment 30 or 31, wherein the reversetranscriptase is a reverse transcriptase with terminal transferaseactivity.

Embodiment 34 is the kit of any one of Embodiments 31-33, wherein thesecond adaptor sequence or the complement thereof is a TSO or acomplement thereof.

Embodiment 35 is the kit of any one of Embodiments 31-34, wherein thekit further comprises an array, wherein the array comprises an attachedcapture probe comprising in a 5′ to a 3′ direction: (i) a spatialbarcode and (ii) a capture domain that binds specifically to the secondadaptor sequence.

Embodiment 36 is a nucleic acid comprising, in the 5′ to 3′ direction: aspatial barcode; a sequence complementary to a second adaptor sequence;a sequence present in a target nucleic acid; and a sequencecomplementary to a first adaptor sequence.

Embodiment 37 is a nucleic acid comprising, in the 3′ to 5′ direction: acomplement of a spatial barcode; a second adaptor sequence; a sequencecomplementary to a sequence present in a target nucleic acid; and afirst adaptor sequence.

Embodiment 38 is the nucleic acid of Embodiment 36 or 37, wherein thesecond adaptor sequence comprises a TSO.

Embodiment 39 is the nucleic acid of Embodiment 36 or 37, wherein thesecond adaptor sequence comprises a complement of a TSO.

Embodiment 40 is the nucleic acid of any one of Embodiments 36-39,wherein the first adaptor sequence is a reverse transcriptase primer.

What is claimed is:
 1. A method of identifying a location of a targetnucleic acid in a permeabilized biological sample, the methodcomprising: (a) generating a cDNA molecule comprising a sequence that issubstantially complementary to the target nucleic acid using a reversetranscription primer comprising (i) a sequence that is substantiallycomplementary to a portion of the target nucleic acid and (ii) a firstadaptor sequence, wherein the step of generating the cDNA moleculeoccurs within the permeabilized biological sample; (b) ligating a secondadaptor sequence to a 3′ end of the cDNA molecule, wherein the step ofligating is performed within the biological sample; (c) releasing thecDNA molecule from the target nucleic acid, such that the cDNA contactsan array, wherein the array comprises an attached capture probecomprising in a 5′ to a 3′ direction: (i) a spatial barcode and (ii) acapture domain that binds specifically to the second adaptor sequenceligated to the cDNA; (d) extending a 3′ end of the capture probe usingthe cDNA as a template; and (e) determining (i) all or a part of thesequence of the target nucleic acid, or a complement thereof, and (ii)all or a part of the sequence of the spatial barcode, or a complementthereof, and using the determined sequences of (i) and (ii) to identifythe location of the target nucleic acid in the permeabilized biologicalsample.
 2. A method of identifying a location of a target nucleic acidin a permeabilized biological sample, the method comprising: (a)generating a cDNA molecule comprising a sequence that is substantiallycomplementary to the target nucleic acid using a reverse transcriptionprimer comprising (i) a sequence that is substantially complementary toa portion of the target nucleic acid and (ii) a first adaptor sequence,wherein the step of generating the cDNA molecule occurs within thepermeabilized biological sample; (b) extending a 3′ end of the cDNAmolecule to include a second adaptor sequence, wherein the step ofextending is performed within the biological sample; (c) releasing thecDNA molecule from the target nucleic acid, such that the cDNA contactsan array, wherein the array comprises an attached capture probecomprising in a 5′ to a 3′ direction: (i) a spatial barcode and (ii) acapture domain that binds specifically to the second adaptor sequence;(d) extending a 3′ end of the capture probe using the cDNA as atemplate; and (e) determining (i) all or a part of the sequence of thetarget nucleic acid, or a complement thereof, and (ii) the sequence ofthe spatial barcode, or a complement thereof, and using the determinedsequences of (i) and (ii) to identify the location of the target nucleicacid in the permeabilized biological sample.
 3. The method claim 1,wherein steps (a) through (c) are performed when the biological sampleis disposed on the array.
 4. The method of claim 1, wherein step (a) isperformed when the biological sample is not disposed on the array andstep (b) is performed when the biological sample is disposed on thearray, and wherein the method further comprises between steps (a) and(b), a step of disposing the biological sample on the array.
 5. Themethod of claim 1, wherein steps (a) and (b) are performed when thebiological sample is not disposed on the array, and wherein the methodfurther comprises between steps (b) and (c), a step of disposing thebiological sample on the array.
 6. The method of claim 1, wherein thesequence that is substantially complementary to a portion of the targetnucleic acid present in the reverse transcription primer comprises apoly(T) sequence.
 7. The method of claim 1, wherein the sequence that issubstantially complementary to a portion of the target nucleic acidpresent in the reverse transcription primer comprises a random sequence.8. The method of claim 1, wherein the second adaptor sequence is atemplate switching oligonucleotide (TSO), or a complement thereof. 9.The method of claim 1, wherein the capture probe further comprises aunique molecular identifier (UMI).
 10. The method of claim 1, whereinthe determining in step (e) comprises sequencing (i) all or a part ofthe sequence of the target nucleic acid, or a complement thereof, and(ii) the sequence of the spatial barcode, or a complement thereof. 11.The method of claim 10, wherein the sequencing is high throughputsequencing.
 12. The method of claim 10, wherein the sequencing issequencing by hybridization.
 13. The method of claim 1, wherein thetarget nucleic acid is RNA.
 14. The method of claim 13, wherein the RNAis an mRNA.
 15. The method of claim 1, wherein the method furthercomprises, prior to step (a), a step of permeabilizing the biologicalsample.
 16. The method of claim 1, wherein the performance of step (a)comprises introducing a reverse transcriptase, dNTPs, and the reversetranscription primer into the permeabilized biological sample.
 17. Anucleic acid comprising, in the 5′ to 3′ direction: a spatial barcode; asequence complementary to a second adaptor sequence; a sequence presentin a target nucleic acid; and a sequence complementary to a firstadaptor sequence.
 18. The nucleic acid of claim 17, wherein the secondadaptor sequence comprises a TSO.
 19. The nucleic acid of claim 17,wherein the second adaptor sequence comprises a complement of a TSO. 20.The nucleic acid of claim 17, wherein the first adaptor sequence is areverse transcription primer.